Over the years many techniques for the genetic transformation of plants have been developed. These methods have as ultimate goal to obtain a transgenic plant, in which all cells contain a foreign DNA comprising a gene of interest (the so-called transgene) stably integrated in their genome, particularly their nuclear genome.
Different plant transformation methods have been described and can be classified into physical DNA delivery methods (e.g. electroporation, PEG-mediated DNA uptake, biolistics) or Agrobacterium-mediated DNA transfer. The latter one frequently is superior in efficiency, simplicity and quality of the transgenic plants (which generally comprise a smaller number of transgenes and have a lower occurence of aberrant transgenes).
Agrobacterium-mediated DNA transformation of plants is based on the capacity of certain Agrobacterium strains to introduce a part of their Ti-plasmid, i.e. the T-DNA, into plant cells and to integrate this T-DNA into the nuclear genome of the cells. It was found that the part of the Ti-plasmid that is transferred and integrated is delineated by specific DNA sequences, the so-called left and right T-DNA border sequences and that the natural T-DNA sequences between these border sequences can be replaced by foreign DNA (European Patent Publication "EP" 116718; 1987 Deblaere et al. (1 987) Meth. Enzymol. 153: 277-293).
Frequently, Agrobacterium-mediated transformation protocols call for the use of readily regenerable e excised plant tissues, organs or parts of organs, such as leaf discs, internodia, stem segments and the like. Alternatively, in vitro cultured tissues (e.g. compact embryogenic callus), suspension cultures or single cells (protoplasts) are employed as starting material to be transformed. For Arabidopsis, protocols have been described wherein seeds or total plants are inoculated (so called in planta transformation protocols).
A common feature of all protocols is that the cells, tissues or plants to be transformed are co-cultivated for a certain time with the Agrobacterium strains and subsequently, the proliferation of the Agrobacterium strains has to be restricted or eliminated by the use of bacteriocides or bacteriostatics such as antibiotics, particularly if further in vitro culturing is required. Frequently, the utilized concentrations of antibiotics interfere with or even inhibit the efficient regeneration of the transformed plant cells. Indeed it has been demonstrated that addition of certain antibiotics with a beta-lactam core structure, such as carbenicillin, to the plant medium may have cytokinin-like effects (WO 97/12512). Furthermore, the Agrobacterium strain of interest frequently comprises a bacterially expressed antibiotic resistance gene, such as but not limited to beta-lactamases, further restricting the spectrum of suitable antibiotics.
A problem frequently observed with the transformation of certain recalcitrant plant species, is that the regenerable cell layer may not be readily accessible to Agrobacterium-mediated transformation. Indeed, histogenetic analysis has demonstrated that e.g., in tomato, shoots induced by cytokinin treatment are derived from a few neocambial cells (Monacelli et al. 1988, Protoplasma 142, 156-163).
Another regularly encountered problem in Agrobacterium mediated transformation is the stress that vigourously proliferating Agrobacterium strains exercise on plants or plant parts, particularly when culturing the transformed plants or tissues in vitro, which may lead to inhibited regeneration or even death of the cells or explants.
Lippincott et al, 1965 (Journal of Bacteriology 90, 1155-1156) describe that auxotrophic mutants of Agrobacterium strain B6 are greatly reduced in infectivity (&lt;1 to 30% of the parent specific infectivity).
Lippincot and Lippincott (1966, Journal of Bacteriology 92, 937-945) describe the further characterization of Agrobacterium mutant strains auxotrophic for adenine, methionine or asparagine and established that infectivity could be increased by simultaneously applying the required nutrient to the infected leaves at the time of infection with the auxotrophic Agrobacteria.
Christen et al. (1984, Z. Pflanzenphysiol. Bd., 113 S. 213-221) demonstrated that leucine- and histidine requiring Agrobacterium tumefaciens mutants grow extremely poorly in the presence of dividing Nicotiana tabacum cv. xanthi protoplasts and suggested the use of such auxotrophic strains for cocultivation with plant protoplasts in the presence of a limiting amount of the required nutrient to allow temporary Agrobacterium division, as an alternative to the use of antibiotics.
Chang et al. (1994, The Plant Journal 5, 551-558) describe a transformation protocol for Arabidopsis involving severing of apical shoots at their bases, inoculation with Agrobacterium at the severed sites, and in planta generation of shoots from the severed sites to produce stably transformed progeny.
An improved in planta transformation method for Arabidopsis was described by Bechtold et al. (1993, C.R. Acad. Sci. Paris, Sciences de la vie 316: 1194-9) based on vacuum infiltration of a suspension of Agrobacterium cells into Arabidopsis plants, followed by selection of transformed progeny in the Ti seed. The authors assumed that a main limiting factor for the transformation frequency would be the restricted persistence of bacteria in the plant from the germination stage until the seed formation.